Several classes of side-chain altered methotrexate (MTX) derivatives will be studied with the aim of obtaining novel antifolates with favorably modified biochemical, pharmacological, and therapeutic properties. Included in the program will be (a) lipophilic ester derivatives whose passive diffusion into cells offers a potential means of treatment of tumors that are MTX-resistant by virtue of impaired active transport, and whose nonpolar character increases their chance of being effective against tumors of the CNS; (b) amide and peptide "prodrugs" that can undergo regionally selective biotransformation to MTX, either in specialized organs such as the liver or in the vicinity of tumors known to secrete amidases or peptidases; and (c) analogues of MTX in which the L-glutamate moiety has been replaced by other amino acids whose structure is designed to promote uptake into cells or bring about tighter binding to the target enzyme dihydrofolate reductase (DHFR). Test systems to be used in evaluating the new compounds synthesized during this program will include spectrophotometric enzyme inhibition assays using DHFR from normal liver and from tumor cells; measurement of (3H)-deoxyuridine and (3H)-thymidine incorporation into DNA and into the acid-soluble nucleotide pool; cytotoxicity assays using MTX-sensitive and MTX-resistant tumor cells in culture; antitumor assays against a variety of experimental tumor systems in animals; and distribution, biotransformation, and pharmacokinetic studies in various species such as mouse, dog, and monkey. Ultimately we hope to select a single compound whose biochemical and pharmacologic properties make it a logical candidate for clinical use. Once this has been done, appropriate pre-clinical studies will be performed and an IND application for a Phase I evaluation in man will be filed.